Characterizing parasitic nematode faunas in faeces and soil using DNA metabarcoding

Abstract

Background Gastrointestinal parasitic nematodes can impact fecundity, development, behaviour, and survival in wild vertebrate populations. Conventional monitoring of gastrointestinal parasitic nematodes in wild populations involves morphological identification of eggs, larvae, and adults from faeces or intestinal samples. Adult worms are typically required for species-level identification, meaning intestinal material from dead animals is needed to characterize the nematode community with high taxonomic resolution. DNA metabarcoding of environmental samples is increasingly used for time- and cost-effective, high-throughput biodiversity monitoring of small-bodied organisms, including parasite communities. Here, we evaluate the potential of DNA metabarcoding of faeces and soil samples for non-invasive monitoring of gastrointestinal parasitic nematode communities in a wild ruminant population. Methods Faeces and intestines were collected from a population of wild reindeer, and soil was collected both from areas showing signs of animal congregation, as well as areas with no signs of animal activity. Gastrointestinal parasitic nematode faunas were characterized using traditional morphological methods that involve flotation and sedimentation steps to concentrate nematode biomass, as well as using DNA metabarcoding. DNA metabarcoding was conducted on bulk samples, in addition to samples having undergone sedimentation and flotation treatments. Results DNA metabarcoding and morphological approaches were largely congruent, recovering similar nematode faunas from all samples. However, metabarcoding provided higher-resolution taxonomic data than morphological identification in both faeces and soil samples. Although concentration of nematode biomass by sedimentation or flotation prior to DNA metabarcoding reduced non-target amplification and increased the diversity of sequence variants recovered from each sample, the pretreatments did not improve species detection rates in soil and faeces samples. Conclusions DNA metabarcoding of bulk faeces samples is a non-invasive, time- and cost-effective method for assessing parasitic nematode populations that provides data with comparable taxonomic resolution to morphological methods that depend on parasitological investigations of dead animals. The successful detection of parasitic gastrointestinal nematodes from soils demonstrates the utility of this approach for mapping distribution and occurrences of the free-living stages of gastrointestinal parasitic nematodes. Graphical abstract

Sensitive and accurate DNA metabarcoding of parasitic helminth mock communities using the mitochondrial rRNA genes

“…Also, DNA extraction is commonly performed using commercial kits, with varied kit performance depending on the sample type. Waeyenberge et al (2019) evaluated 15 DNA extraction procedures in nematode mixtures and found varying degrees of DNA yield 25 . Additionally, replicate sampling can be performed on a particular community to detect as many species as possible 49 , 50 .…”

“…Its specificity remains an issue as studies have revealed that only a small proportion of sequence reads are specific to nematodes 22 , 23 . In addition to environmental samples, the non-invasive sampling of gastrointestinal nematodes in animal hosts through fecal samples emphasizes the potential of helminth DNA metabarcoding 24 , 25 . Studies have investigated the impact of routine anti-parasitic treatment on parasitic nematode communities of cattle, illustrating the power of DNA metabarcoding in monitoring anti-parasitic resistance in livestock 26 .…”

Parasite Spillover from Domestic Sheep to Wild Reindeer—The Role of Salt Licks

“…To examine the genetic diversity of N. battus isolated from sheep and reindeer, we retrieved the ITS sequences from NCBI non-redundant nucleotide databases, including 8 N. battus worms isolated from sheep and reindeer in the focal areas as described by Davey et al [ 14 ] ( Table 1 ). Percentage identity was compared pairwise between the sequences using the pid function in the Biostrings package in R [ 15 ], and a sequence alignment of these and the 11 most frequent N. battus amplicon sequence variants generated from amplicon sequencing of soil samples.…”

Soil-Borne Nematodes: Impact in Agriculture and Livestock and Sustainable Strategies of Prevention and Control with Special Reference to the Use of Nematode Natural Enemies

“…However, advanced molecular methods, e.g., qPCR followed by a high resolution melting analysis of ITS-1, open other convenient, rapid, and reliable alternative methods for taxonomic affiliation [81]. Similarly, new molecular tools, e.g., DNA metabarcoding using only faecal samples, have been claimed to provide a non-invasive method for assessing parasitic nematode populations [82]. Nevertheless, due to the high cost and the relatively small amplicon length, this cannot be considered a cost-effect method.…”